Fluorescent detection of reaction products is common in a number of analytical settings. Typically, analytical instruments for monitoring fluorescent reactions are equipped with reaction chambers designed to minimize flourescence from external sources, for example, by providing a light-impermeable compartment constructed of non-fluorogenic materials. However, such precautions may not prevent fluorescent emissions from contamination sources, such as flecks of hair or skin introduced to the chamber during sample manipulation.
The present invention provides a sample tube which is constructed to reduce fluorescence from external sources. The tube is particularly usefull in nucleic acid amplification reactions, such as the polymerase chain reaction (PCR) in which progress of the reaction is monitored.
PCR has become a research tool of major importance with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like, e.g. Arnheim et al (cited above); Gilliland et al, Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan et al, PCR Methods and Applications, 1: 222-228 (1992); Green et al, PCR Methods and Applications, 1: 77-90 (1991); Blackwell et al, Science, 250: 1104-1110 (1990).
While a number of instruments have been developed for carrying out nucleic acid amplification, most employ basic PCR technology, e.g. Johnson et al, U.S. Pat. No. 5,038,852 (computer-controlled thermal cycler); Wittwer et al, Nucleic Acids Research, 17: 43534357 (1989)(capillary tube PCR); Hallsby, U.S. Pat. No. 5,187,084 (air-based temperature control); Garner et al, Biotechniques, 14: 112-115 (1993)(high-throughput PCR in 864-well plates); Wilding et al, International application No. PCT/US93/04039 (PCR in micro-machined structures); Schnipelsky et al, European patent application No. 90301061.9 (publ. No. 0381501 A2)(disposable, single use PCR device), and the like. Important design goals fundamental to PCR instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-PCR processing steps, high speed cycling, minimization of sample volumes, real time measurement of amplification products, minimization of cross-contamination, or sample carryover, and the like.
Recently, PCR designs have focused on instruments that permit the amplification reaction to be carried out in closed reaction chambers and monitored in real time. Closed reaction chambers are desirable for preventing cross-contamination, e.g. Higuchi et al, Biotechnology, 10: 413417 (1992} and 11: 1026-1030 (1993) and Holland et al, Proc. Natl. Acad. Sci., 88: 72767280 (1991). Real time monitoring is particularly desirable in the analysis of diagnostic samples, where high frequencies of false positives and false negatives can severely reduce the value of the PCR-based procedure.
Moreover, real time monitoring of PCR permits far more accurate quantitation of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the PCR. Real time monitoring also permits the efficiency of the PCR to be evaluated, which can indicate whether PCR inhibitors are present in a sample.
Holland et al (cited above) and others have proposed fluorescence-based approaches to provide real time measurements of amplification products during PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present, or they have employed probes containing fluorescer-quencher pairs (the so-called "Tac-Man" approach) that are cleaved during amplification to release a fluorescent product whose concentration is proportional to the amount of double stranded DNA present.
Unfortunately, successful implementation of these approaches has been impeded because the required fluorescent measurements must be made against a very high fluorescent background. Thus, even minor sources of instrumental noise, such as the formation of condensation in the chamber during heating and cooling cycles, formation of bubbles in an optical path, particles or debris in solution, differences in sample volumes--and hence, differences in signal emission and absorbence, and the like, have hampered the reliable measurement of the fluorescent signals.
Parent U.S. patent application Ser. No. 08/235,411, abandoned, describes an apparatus that provides stable and reliable real time measurement of fluorescent indicators of amplification products resulting from any of the available nucleic acid amplification schemes. This apparatus operates by directing into a fluorescent mixture an excitation beam having appropriate energy to excite the fluorescent centers present in the mixture. The present invention is directed to an improvement of this apparatus which includes using a reaction tube that reduces background fluorescence measured from a test sample by reducing the amount of exogenous flourescence that enters the tube from outside sources such as contamination present in the apparatus tube-holder. As particularly described herein, the tube is a plastic consumable tube having an irregular or roughened outer surface that deflects or diffuses incident fluorescence emissions emanating from outside the tube. However, it is also preferable that the tube remain sufficiently clear to permit the human user to visualize fluid volume contained in the tube. Moreover, the tube may be sealable and provide a limited transparent window region to allow transmittance of an excitation beam to a sample held in the tube.
While the described tube is particularly suited for use in a PCR monitoring apparatus, such as that described in U.S. patent application Ser. No. 08/235,411, abandoned, it can be appreciated that such tubes are also suited for use in other instruments in which detection of light emissions is measured, and particularly those in which such measurement is carried out in a "plate-reader" format.